NDT Advance Access published online on January 11, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfm789
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Transforming growth factor-β-induced alpha-smooth muscle cell actin expression in renal proximal tubular cells is regulated by p38β mitogen-activated protein kinase, extracellular signal-regulated protein kinase1,2 and the Smad signalling during epithelial–myofibroblast transdifferentiation
1 Department of Pathophysiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary 2 Hungarian Academy of Sciences, Semmelweis University Research Group for Pediatrics and Nephrology, Budapest, Hungary 3 Department of Dermatology, Department of Medical Biochemistry and Molecular Biology, and MediCity Research Laboratory, University of Turku, Finland, 4 1st Department of Medicine, Faculty of Medicine, Semmelweis University, Budapest, Hungary
Correspondence and offprint requests to: Istvan Mucsi, 1st Department of Medicine, Faculty of Medicine, Semmelweis University, Budapest, 2/A Koranyi S. u, Budapest, H-1083, Hungary. Tel: + 36-20-825-8671; Fax: + 36-1-210-1220; E-mail: istvan{at}nefros.net
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Abstract |
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Background. Transforming growth factor-β (TGFβ)-induced epithelial–myofibroblast transdifferentiation is a central mechanism contributing to the pathogenesis of progressive tubulo-interstitial fibrosis. We wanted to dissect the role of extracellular signal-regulated protein kinase (ERK1,2), p38 mitogen-activated protein kinase (p38 MAPK) and the receptor-regulated Smad proteins in the regulation of
-smooth muscle cell actin (SMA) expression, a hallmark of myofibroblast formation, induced by TGFβ in renal proximal tubular cells.
Methods. Activation of signalling molecules was assessed by western blotting using phospho-specific antibodies. To specifically interfere with signalling cascades, porcine proximal tubular cells (LLC-PK/AT1) were infected with recombinant replication-deficient adenoviruses. In other experiments, specific kinase inhibitors were used. The SMA synthesis was assessed by western blotting or immunofluorescent staining of cellular SMA. To assess the regulation of the SMA promoter, tubular cells were transiently transfected with a 785 bp SMA promoter–luciferase reporter construct and vectors interfering with the Smad pathway.
Results. Blocking ERK1,2 activation with PD98059 or p38 MAPK with SB 203580 potently inhibited the TGFβ-induced SMA synthesis in renal tubular cells. Adenoviral expression of dominant negative (DN) p38β but not of p38 potently inhibited SMA expression. Furthermore, adenoviral expression of DN MKK6b but not of DN MKK3b caused a substantial inhibition of the TGFβ effect, confirming the role of p38β in the regulation of TGFβ-induced SMA expression. Finally, inhibiting the Smad pathway with adenovirally delivered Smad7 and DN Smad3 also blocked TGFβ-induced SMA synthesis.
Conclusion. TGFβ-induced SMA expression is regulated by the coordinated activation of a complex system of parallel MAPK and Smad signalling pathways in renal proximal tubular cells during epithelial–mesenchymal transdifferentiation.
Keywords: alpha-smooth muscle cell actin; epithelial–myofibroblast transdifferentiation; p38 nitrogen-activated protein kinase; renal proximal tubular cell; Smad proteins
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Introduction |
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Epithelial–mesenchymal transdifferentiation (EMT)—a process known to play an important role during organ development, wound healing and tumour invasion—has recently emerged as a central mechanism in the pathogenesis of tubulo-interstitial fibrosis (TIF) [1–4]. Pro-fibrotic stimuli induce a coordinated genetic program in the highly differentiated tubular epithelial cells (TEC) that results in profound phenotypic changes [5]. This process is associated with a marked reorganization of the cytoskeleton. Transformed cells become motile and migrate into the interstitium. Some of the transdifferentiating cells express
-smooth muscle cell actin (SMA) that is completely absent in tubular epithelial cells. The appearance of SMA in the transdifferentiating cells during EMT is a hallmark of myofibroblast (MF) formation. MFs are thought to play a central role in excessive matrix deposition during the fibrotic process [6,7]. However, SMA is more than a marker of this process, as it is instrumental for MF contraction and force generation for tissue retraction—an important feature of the fibrotic process [6,7]. Transforming growth factor-β (TGFβ) is a central regulator of the pathogenesis of TIF and it is also one of the most potent growth factors to induce EMT in tubular cells [8]. TGFβ induces EMT through a complex interplay of signalling pathways, the exact details of which have still not been fully elucidated. In different cellular systems, the Smad proteins [9,10], the Rho-family GTPases [11,12], the extracellular signal-regulated protein kinase (ERK1,2) [5,13] and the p38 mitogen-activated protein kinase (MAPK) [14] have all been shown to contribute to the induction of EMT by TGFβ.
The p38 MAPK family consists of four isoforms: p38, p38β, p38
and p38
[15]. Recently, it has been demonstrated that different p38 isoforms may have different functions [16]. It is currently not known which p38 isoform is involved in the regulation of TGFβ-induced EMT in renal tubular cells. This could potentially have important implications as the different isoforms can specifically be inhibited using newly developed isoform-specific inhibitors.
Several studies have demonstrated that TGFβ induces de novo SMA synthesis in renal tubular cells during EMT [12,17]. SMA expression by TGFβ is regulated at least in part at the transcriptional level. Regulation of the SMA transcription is remarkably complex and shows substantial tissue specificity. In different cellular systems, both the Smad proteins and members of the MAPK family are known to regulate SMA transcription [9,18].
We have previously shown, using LLC-PK1 porcine proximal tubular cells, that several signalling systems were indispensable for the TGFβ and cell-contact disassembly-induced EMT [12,19]. Here we report results of experiments that were designed to study the role of ERK1,2, p38 MAPK and the receptor-regulated Smad proteins (R-Smad) in the SMA protein expression induced by TGFβ using LLC-PK1 porcine renal proximal tubular cells, the same cellular model system that we had used in our earlier work. The SMA in LLC-PK1 cells was first seen after 24 h of incubation with TGFβ, with a further increase during the subsequent 72 h. TGFβ induced a biphasic activation of ERK1,2 and p38, as well as Smad2 and Smad3. Inhibition of both ERK1,2 and p38β MAPK blocked the effect of TGFβ on SMA protein synthesis. Furthermore, inhibition of Smad signalling with Smad7 or dominant negative (DN) Smad3 also inhibited TGFβ-induced SMA protein expression in tubular cells. Together, these observations provide evidence for the coordinated role of MAPK and Smad signalling pathways in mediating the phenotypic alterations of renal tubular cells in response to TGFβ.
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Material and methods |
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Cell culture
Porcine proximal tubular epithelial cells stably expressing the rabbit AT1 receptor (LLC-PK1/AT1) were grown in Dulbecco's modified Eagle's medium (DMEM, Sigma, St Louis, MO, USA) supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU/ml penicillin G and 100 µg/ml streptomycin in a humidified incubator at 37°C and 5% CO2.
Reagents and antibodies
Human recombinant TGFβ1 was obtained from Sigma (St Louis, MO, USA), and the p38 inhibitor SB203580 and the MEK inhibitor PD98059 were from Calbiochem (San Diego, CA, USA).
Anti-SMA (1A4) and anti-FLAG antibodies were purchased from Sigma. Anti-phospho-p38, anti-p38, anti-phospho-ERK1/2 and anti-ERK1/2 antibodies were from Cell Signaling Technology (Beverly, MA, USA). Antisera against phospho-Smad2 (PS2) and phospho-Smad1 (PS1), which show cross-reactivity with phosphorylated Smad3, were kind gifts from Dr A. Moustakas (Ludwig Institute for Cancer Research, Uppsala, Sweden) [10]. Polyclonal Smad2 and Smad3 antibodies were from Zymed Laboratories Inc. (San Francisco, CA, USA), and rat monoclonal anti-HA 3F10 antibody was from Roche Molecular Biochemicals, Mannheim, Germany. Anti-mouse antibody was obtained from Amersham Biosciences (Uppsala, Sweden), anti-rabbit IgG was from DAKO, Glostrup, Denmark and anti-rat antibody was from Santa Cruz Biotechnology (San Francisco, CA, USA). The antibodies for immunostaining anti-rat-rhodamine and anti-mouse IgG, heavy and light chain fluorescein conjugates were from Calbiochem.
Plasmids and transient transfection
The PA3-Luc vector containing a 765 bp fragment of the rat SMA promoter was a kind gift from Dr R.A. Nemenoff (Department of Medicine, University of Colorado, CO, USA). The pSmad7 expression construct was a kind gift from Dr E.P. Böttinger (Albert Einstein College of Medicine, Bronx, NY, USA) [20]. The vector encoding DN Smad3 was described and used in our previous work. The thymidine kinase-driven Renilla luciferase vector (pRL-TK; Promega, Madison, WI, USA) was used as an internal control.
LLC-PK1/AT1 cells were grown on six-well plates and transfected at 50% confluence with 2.5 µl FuGene6 (Roche) reagent using 0.5 µg pSMA-Luc, 0.05 µg pRL-TK and 2 µg of interfering plasmid or pcDNA3. Sixteen hours later the cells were washed three times with PBS and the medium was replaced with serum-free DMEM. Six hours later 5 ng/ml TGFβ or its vehiculum was added for 20 h. Cells were then lysed in 500 µl passive lysis buffer (Promega) and the samples were subjected to freezing/thawing. Firefly and Renilla luciferase activities were measured with the Dual Luciferase Reporter Assay Kit (Promega) following the instructions of the manufacturers. Results were normalized to the values obtained by the Renilla measurements for each sample.
Reverse transcription PCR (RT-PCR) analysis of p38 isoform expression [21,22]
Total cellular RNA was extracted from cultured cells using the Qiagen RNeasy Mini kit (Qiagen Nordic, Helsinki, Finland) according to the manufacturer's instructions. The cDNA was synthesized using 1 µg RQ1 RNase-free DNase-treated RNA, M-MLV reverse transcriptase, RNase inhibitor and random hexamer primers (all reagents were from Promega). The final RT reaction mix was 20 µl and the program was as follows: 15 min at 42°C, 5 min at 99°C and 5 min at 5°C.
PCR reaction was carried out by using the DyNAzyme EXT kit (Finnzymes, Espoo, Finland) according to the manufacturer's instructions. Briefly, all samples were prepared by mixing in 50 µl final volume, 5 µl optimized DyNAzyme EXT buffer, 1 µl dNTPs (200 µM), 1 µM forward and reverse primers and 1 µl DyNAzyme (1U). The following program was used: 2 min at 94°C, and then: (30 s at 94°C, 30 s at 55°C and 60 s at 72°C) x 34 times followed by 10 min at 72°C at the end. The sequences of the primers were as follows:
p38: fwd 5'-AAC CTG TCT CCA GTG GGC TCT-3'; rev 3'-CGT AAC CCC GTT TTT GTG TCA-5'; p38β: fwd 5'-CAC CCA GCC CTG AGG TTC T-3'; rev 3'-AGA TGC TGC TCA GGT CCT TCT-5'; p38: fwd 5'-CGC CTC CGG GCT GAG TTT-3'; rev 3'-GCT TGC ATT GGT CAG GAT AGA-5'; p38: fwd 5'-TGC TCG GCC ATC GAC AA-3'; rev 3'-TGG CGA AGA TCT CGG ACT GA-5'; β-actin: fwd 5'-TCA CCC ACA ctg tgc cca tct acg c-3'; rev 3'-cag cgg aac cgc tca ttg cca atg g-5'.
Specificity of amplification was confirmed by running each amplicon on a 3% agarose gel and obtaining a single band of the expected size (p38—70 bp, p38β—98 bp, p38—105 bp and p38—81 bp). The gel contained a final concentration of 0.5 µg/ml ethidium bromide. Expression of β-actin was used as control.
Recombinant adenoviruses
Recombinant replication-deficient adenovirus RAdLacZ, which contains the Escherichia coli β-galactosidase gene under the control of the cytomegalovirus immediate early promoter, was kindly provided by Dr Gavin W.G. Wilkinson (University of Cardiff, Wales, UK). Recombinant adenovirus for DN Smad3 (RAdSmad3DN) [10] and the SMAD7 adenovirus were kindly provided by Dr A. Moustakas (Ludwig Institute for Cancer Research, Uppsala, Sweden), and adenovirus for constitutively active (CA) MEK1 (RAdMEK1CA) was provided by Dr M. Foschi (University of Florence). Adenoviruses for CA MKK3b (RAdMKK3bE), CA MKK6b (RAdMKK6bE), DN p38 (RAdp38AF) and DN p38β (RAdp38βAF), DN MKK3b (RAdMKK3bA) and MKK6b (RAdMKK6bA) were all kindly provided by Dr Jiahua Han (Scripps Research Institute, La Jolla, CA, USA). Replication-deficient (E1- and E3-) adenoviruses RAdSmad2, RAdSmad3 harbouring human Smad2 and Smad3 cDNAs, respectively, with an N-terminal haemagglutinin (HA) tag, were described previously [23].
Infection of cells with recombinant adenoviruses
Cells were infected in suspension with the adenoviruses at 1 MOI in DMEM with 1% FCS, and then plated and incubated for 18 h. Subsequently, the medium was replaced with fresh 1% FCS DMEM. Six hours later, 5 ng/ml TGFβ was added for the time indicated. The cells then were harvested in SDS sample buffer and analysed by western blotting.
Western blotting
LLC-PK1/AT1 cells were plated onto 6-cm dishes and grown until confluent. After washing the cells with PBS, the medium was replaced with serum-free DMEM for 24 h. Subsequently 5 ng/ml TGFβ was added. In some experiments, the cells were pretreated with 1 and 5 µM SB203580 and 40 µM PD98059. At the time indicated, cells were harvested into SDS sample buffer.
Cell lysates were fractionated on 12% SDS–polyacrylamide gel and transferred to the Hybond ECL nitrocellulose membrane (Amersham Biosciences). The membranes were blocked with 5% skim milk. After ample washing, specific primary antibodies and subsequently peroxidase-conjugated secondary antibodies were added. The blots were visualized by the ECL detection system (Amersham Biosciences).
Immunostaining
Cells were pretreated with SB203580 and PD98059, and treated with TGFβ. After 4 days of incubation, medium was removed, the cells were washed with ice-cold PBS, fixed with –20°C methanol and incubated at –20°C for 6 min. After washing in PBS, the cells were stained with the appropriate primary and secondary antibodies, and Hoechst reagent. The results were visualized by a Leica fluorescence microscope.
Statistical analysis
Experiments were repeated at least three times. For western blot and immunostaining experiments, representative images are shown. Transfections were performed in duplicates. The results are shown as mean ± SD and expressed as a ratio of the luciferase activity in the TGFβ-treated groups compared to the vehicle-treated groups (fold stimulation). One-way ANOVA was used to analyse the data.
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Results |
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TGFβ activates multiple signalling pathways in LLC-PK1 tubular cells
In different cellular systems, both ERK1,2, p38 MAPK and the Smad proteins were shown to contribute to the induction of EMT by TGFβ. In our proximal tubular cells, TGFβ induced phosphorylation, i.e. activation of both ERK1,2 and p38 MAPK (Figure 1A). The phosphorylation of these proteins was somewhat delayed, especially for p38 MAPK, for which increased phosphorylation was first seen at 60 min after stimulation. Furthermore, the initial activation of p38 was transient, the activity declining close to baseline within 2 h. In contrast, increased phosphorylation of ERK1,2 was more prolonged, the phosphorylation remaining increased throughout the first 3 h of incubation.
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Interestingly, phosphorylation, that is, activation of Smad2 and Smad3, was more rapid (Figure 1A). For both these proteins, an increased phosphorylation was seen as early as 15 min after the addition of TGFβ to the medium, and the maximum effect was seen at 60 min. This effect was transient for both proteins, with a more rapid decline of the phosphorylation for Smad3.
TGFβ induces a biphasic increase in MAPK and Smad phosphorylation in LLC-PK1 cells
Expression of SMA was not seen until 24–48 h of TGFβ exposure and reached a maximum after 3–4 days of incubation (not shown). Therefore, we analysed the effect of prolonged (up to 4 days) TGFβ treatment on R-Smads and MAP kinases in LLCPK1/AT1 cells (Figure 1B). TGFβ activated these signalling proteins with a markedly different time course. Activation of Smad3 was quite transient, with essentially no further activation seen after 4 h of stimulation with TGFβ. On the other hand, the phosphorylation of ERK1,2, p38 MAPK and Smad2 followed a biphasic pattern. After the initial peak at 1 h, the activation returned towards baseline for all three proteins but began to rise again thereafter. For Smad2 and ERK1,2, the phosphorylation remained significantly increased even during the intermediate period and started to increase again after 12–24 h of TGFβ incubation. The phosphorylation of these signalling proteins remained increased until the end of the 4-day incubation period. For p38 MAPK, however, the activation returned close to the baseline by the fourth hour and the second peak started quite late at 24–48 h (Figure 1B). The phosphorylation level of these signalling molecules remained at or below baseline during the incubation period in quiescent control cells without TGFβ stimulation (not shown).
Both ERK1,2 and p38 MAPKs contribute to the TGFβ-induced SMA synthesis in renal tubular cells
To assess the functional significance of the ERK1,2 signalling pathway in TGFβ-induced SMA synthesis, the specific MEK1 inhibitor PD98059 was used. Alpha SMA synthesis was assessed by western blotting and immunofluorescent staining of cellular SMA. Treatment of cells with TGFβ for 96 h resulted in the induction of SMA (Figure 2A) and in marked alteration in the morphology of the cells (Figure 2B). Pretreatment of the cells with PD98059 potently inhibited the TGFβ-induced SMA synthesis (Figure 2A) and also blocked the formation of actin fibres (Figure 2B).
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Treatment of the cells with SB 203580, a specific inhibitor of p38 MAPK, also inhibited
SMA protein expression. This effect was already seen with a concentration as low as 1 µM, and 5 µM SB203580 blocked the TGFβ effect on SMA protein expression completely (Figure 2A). Furthermore, SB203580 also prevented the incorporation of SMA into the thick actin fibres that were formed upon TGFβ treatment (Figure 2B).
TGFβ activates SMA expression through MKK6 –p38β
As SB203580 is reported to inhibit the activity of p38 and p38β but not p38 or p38 isoforms [24], (therefore, we wanted to determine whether both the and β isoforms played a role in the TGFβ-induced SMA expression. To this end, LLC-PK/AT1 cells were infected with replication-deficient adenoviral vectors expressing the DN mutant of p38 and p38β (Figure 3A). A 4-day incubation in the presence of TGFβ induced marked SMA protein expression in tubular cells infected with the control adenovirus, RAdLacZ. Dominant inhibitory p38 caused a slight decrease in the effect of TGFβ, whereas adenoviral expression of DN p38β inhibited SMA expression almost completely. No further increase in the inhibitory effect of DN p38β was seen when the cells were infected with both DN p38 and DN p38β together (Figure 3A).
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Activation of the p38 MAPK occurs through phosphorylation by specific upstream protein kinase kinases, namely MKK3 and MKK6. MKK3 activates p38
, p38 and possibly p38; however, MKK6 activates all four isoforms. To explore the role of these kinases in the increased SMA expression, we also exploited adenoviral gene delivery of mutated signalling molecules. Infection of the cells with a vector harbouring DN MKK6b caused a substantial inhibition of the TGFβ effect (Figure 3A). On the other hand, DN MKK3b, which is expected to inhibit all p38 MAPK isoforms except p38β, had no significant effect on the increased SMA expression induced by TGFβ. These results together point towards the predominant contribution of p38β to the TGFβ-induced SMA expression. In concert with the above results, we detected the expression of both p38β and p38 but not p38 or p38 in LLC-PK1 cells using RT-PCR analysis (Figure 3B).
To test the effect of prolonged activation of ERK1,2 and p38 MAPK on SMA protein expression, tubular cells were infected with CA MAP kinase kinases (Figure 4). Active MEK1 induced a marked phosphorylation of ERK1,2 but not of p38 MAPK. In cells infected with CA MKK3, a modest phosphorylation of p38 MAPK was seen, possibly corresponding to the presence of the p38 isoform. CA MKK6, on the other hand, induced a robust phosphorylation of the endogenous p38 MAPK, suggesting the more significant contribution of the β isoform. None of the CA MAP kinase kinases, however, were able to reproduce the effect of TGFβ on SMA synthesis. To confirm this finding, we co-infected tubular cells with both CA MAP kinase kinases and wild-type (WT) p38 or p38β. When CA MKK3 and WT p38 were co-infected, a robust activation of p38 MAPK was seen. However, co-infection of MKK3 and p38β together resulted only in a minimal phosphorylation of both endogenous and the recombinant p38 MAPK, confirming the specificity of MKK3 in activating p38 predominantly. CA MKK6, on the other hand, readily activated both p38 and p38β. In spite of the robust phosphorylation of the recombinant p38 MAP kinases in this experiment, SMA expression was not seen suggesting that activation of p38 MAPK is necessary but not sufficient to induce SMA expression in tubular cells.
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The receptor-regulated Smad proteins are involved in the regulation of the TGFβ-induced
SMA protein expression in renal tubular cellsTo assess if the R-Smads, Smad2 or Smad3, contribute to the effect of TGFβ on
SMA protein expression, multiple experimental approaches were utilized. Smad transcriptional activity can specifically be blocked by using dominant inhibitory or DN mutants of the R-Smad proteins. Furthermore, Smad7 also inhibits Smad signalling by preventing the association of the R-Smads with the active type I receptor. First we used transient transfection experiments in which LLC-PK1/AT1 cells were co-transfected with the 785 bp SMA promoter–luciferase reporter construct and expression vectors encoding Smad7 or DN Smad3, respectively. As shown in Figure 5A, expression of both Smad7 and DN Smad3 inhibited the effect of the cytokine on the SMA promoter construct.
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To confirm the effect of inhibition of Smad signalling on TGFβ-induced
SMA protein synthesis, tubular cells were infected with adenoviral expression vectors encoding Smad7 or DN Smad3, respectively. Over-expression of both Smad7 and DN Smad3 almost completely blocked the TGFβ-induced SMA protein synthesis in proximal tubular cells (Figure 5B) confirming the proposed role of Smad signalling in this effect. |
Discussion |
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In this paper we have shown that in renal proximal tubular cells, both ERK1,2, p38 MAPK and the R-Smads are required for the induction of
SMA expression by TGFβ, a hallmark component of epithelial–myofibroblast transdifferentiation. Furthermore, we have shown for the first time that p38 MAPK is primarily activated via the MKK6–p38β cascade, and the other p38 isoforms are unlikely to contribute significantly to the transcriptional regulation of SMA by TGFβ in this system. Blocking ERK1,2 or p38β by specific kinase inhibitors or by adenoviral delivery of interfering mutants of the p38 signalling cascade, largely prevented SMA synthesis and the incorporation of SMA into actin fibres upon TGFβ stimulation. Similarly, inhibition of the Smad signalling system also blocked SMA expression in our tubular cells.
These results contribute to accumulating evidence obtained in different model systems of TGFβ-induced EMT that suggest that several parallel signalling systems cooperate in order to execute the complex cellular program of EMT. A microarray-based screen of transcriptional profiles of TGFβ-induced EMT in human keratinocytes revealed that different cellular ‘response modules’ are regulated by different signalling cascades. For example, ERK1,2 plays an important role in TGFβ-directed cell motility and disruption of adherens junctions, whereas the Smad pathway regulates the expression of several mesenchymal markers [5]. Our results, similar to recent reports [25], demonstrate that parallel signalling pathways are also involved in regulating single components of EMT, such as SMA expression.
ERK1,2 activation has been shown to contribute to TGFβ-induced EMT in different epithelial cells, including renal tubular cells [13]. TGFβ-induced SMA protein synthesis was blocked by the specific MEK1 inhibitor PD98059 in our renal tubular cells, as well. Blocking ERK1,2 activation also prevented the appearance of cellular SMA staining, demonstrating the importance of the ERK1,2 cascade in the regulation of epithelial–mesenchymal transition. Interestingly, prolonged activation of the ERK1,2 cascade by infecting tubular cells with adenoviral vectors expressing CA MEK1 mutant did not induce SMA expression, suggesting that activation of ERK1,2 alone is not sufficient to trigger the complex cellular program. Alternatively, we cannot rule out the possibility that the abiphasic activation pattern is necessary for the specific cellular response to ERK1,2 activation and this is not well simulated by the prolonged ERK1,2 activation induced by the mutant MEK1.
We have shown here that TGFβ induced phosphorylation of p38 MAPK. Furthermore, blocking p38 MAPK by the specific inhibitor SB 203580 inhibited SMA protein synthesis and actin fibre formation induced by TGFβ. Also, p38 MAPK is readily activated by TGFβ in different cells and it is thought to mediate important Smad-independent TGFβ responses. In addition, p38 has been shown to contribute to the regulation of SMA in smooth muscle cells [18]. Furthermore, p38 MAPK has been shown to contribute to the regulation of EMT in different cells [10,14]. Recently p38 MAPK has been implicated in TGFβ-induced EMT in renal tubular cells [13]. The molecular details of how TGFβ-induced p38 MAPK could potentially regulate EMT or SMA expression in renal tubular cells, however, have not yet been elucidated.
The signalling specificity of p38 MAPK may, at least in part, be determined by the specific isoforms expressed and/or activated in a given cell type [16]. Our results suggest that p38β and p38, but not p38 or p38, are expressed in our renal tubular cells. Using adenoviral gene delivery, we have demonstrated that TGFβ induces SMA expression primarily via the MKK6–p38β pathway. Bhowmick et al. have also implicated p38β in the regulation of EMT induced by TGFβ in NMuMG epithelial cells [14]. It has also been suggested that this isoform may inhibit apoptosis, whereas p38 facilitates programmed cell death in tumour cells [26]. Based on this information, one could postulate that the expression pattern of the p38 MAPK isoforms could possibly determine if TGFβ-induced p38 activation results in apoptosis or in a different set of cellular responses, including EMT, in epithelial cells.
The Smad family of signalling proteins has also been implicated in the regulation of TGFβ-induced EMT [8,10]. Our results suggest that in renal tubular cells, both Smad2 and Smad3 contribute to SMA expression induced by TGFβ. Blocking R-Smad activation by over-expressing Smad7 or Smad3 activation using DN Smad3 inhibited SMA synthesis almost completely. Similar to our findings, Valcourt et al. have recently presented data suggesting that both Smad2 and Smad3 are required for TGFβ-induced EMT in human and mouse epithelial cells [10]. Furthermore, Phanish et al. have demonstrated that the TGFβ-induced up-regulation of SMA expression was dependent on both R-Smad proteins in transformed proximal tubular cells [9].
Previous studies have shown that the first 125 base pairs of the SMA promoter drive maximal reporter expression in smooth muscle cells. This region contains two CC(A/T)richGG cis-acting elements and a recently described TGFβ control element (TCE). The CArG motifs are binding sites for the transcriptional activator serum response factor (SRF), whereas the TCE likely interacts with Kruppel factor-like transactivators. Earlier we found that TGFβ inducibility of the SMA promoter in our tubular cells showed similar structural requirements [12]. Recently, Hu et al. described a Smad-binding element (SBE) [27] at –522 to –513 from the transcriptional start site. It is possible that this element contributes to the observed effect of TGFβ on SMA expression in renal tubular cells. Further studies are needed, however, to establish this possibility.
Inhibition of both ERK1,2, p38 MAPK, and the Smad pathway resulted in an almost complete blockage of SMA synthesis in our experiments. On the other hand, isolated and prolonged activation of neither ERK1,2 nor p38 was sufficient to induce SMA expression. Our experiments were not designed to study the complex organizational pattern of these individual signalling pathways; however, we speculate that these parallel pathways cooperate to regulate the activity and/or the expression of downstream effectors. Multiple mechanisms for crosstalk between ERK1,2, p38 MAPK, and the Smad pathway have been described. Both ERK1,2 [28] and p38 [23] have been reported to contribute to Smad activation. In our experiments, Smad phosphorylation preceded both ERK1,2 and p38 MAPK activation. Furthermore, MAPK inhibitors did not block the TGFβ-induced Smad phosphorylation (not shown). Previous reports, including work by Leivonen and Kähäri, have demonstrated that the p38 MAPK [29–31] cascades can be activated via Smad-dependent mechanisms. We are not aware of findings suggesting Smad-dependent ERK activation. On the other hand, we did not see reduced MAPK activation in cells overexpressing Smad7, the inhibitory Smad protein. These preliminary results together provide argument against a hierarchical model of these signalling cascades and we propose that these three pathways are organized to cooperate in a parallel system. Further work, however, is needed to dissect this important aspect of this complex signalling system.
Better understanding of the details of the complex signalling systems regulating EMT may offer opportunities for targeted pharmacological interventions to halt this process that is one of the core mechanisms of tubulo-interstitial fibrosis. Dissecting the specific role played by different p38 MAPK isoforms may be of clinical significance in this respect as isoform-specific p38 inhibitors are already being designed for potential use under different clinical conditions.
In summary, we report here that both ERK1,2, p38β MAPK, and Smad2 and Smad3 are required for the TGFβ-induced induction of SMA expression in renal proximal tubular cells during EMT. Based on our results, we suggest that the cooperation of parallel signalling pathways is necessary to elicit this cellular response. Further experiments to elucidate the molecular mechanisms involved in the transcriptional regulation of SMA during TGFβ-induced EMT in renal proximal tubular cells are under way in our laboratories.
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Acknowledgments |
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This work was supported by grants from: OTKA (T042651, AT 048767), ETT 104/2006, Hungarian Kidney Foundation, TéT-CIMO (SF-11/01), the Academy of Finland, Sigrid Juselius Foundation, Finnish Cancer Research Foundation and from the Turku University Central Hospital (EVO grant 13336). I.M. is a Békésy Postdoctoral Fellow of the Hungarian Ministry of Education. Part of this work was presented at the ERA/EDTA Congress in Berlin in 2003. We appreciate the skillful technical assistance by Sarolta Adamko, Marjo Hakkarainen, Sari Pitkänen and Johanna Markola.
Conflict of interest statement. None declared.
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Notes |
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5 Present address: VTT Medical Biotechnology, FI-20520 Turku, Finland.
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References |
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|
TopAbstractIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
- Zeisberg M, Kalluri R. The role of epithelial-to-mesenchymal transition in renal fibrosis. J Mol Med (2004) 82:175–181.[CrossRef][Web of Science][Medline]
- Strutz F, Okada H, Lo CW, et al. Identification and characterization of a fibroblast marker: FSP1. J Cell Biol (1995) 130:393–405.
[Abstract/Free Full Text] - Strutz F, Muller GA. Renal fibrosis and the origin of the renal fibroblast. Nephrol Dial Transplant (2006) 21:3368–3370.
[Free Full Text] - Slattery C, Campbell E, McMorrow T, et al. Cyclosporine A-induced renal fibrosis: a role for epithelial–mesenchymal transition. Am J Pathol (2005) 167:395–407.
[Abstract/Free Full Text] - Zavadil J, Bitzer M, Liang D, et al. Genetic programs of epithelial cell plasticity directed by transforming growth factor-b. Proc Natl Acad Sci USA (2001) 98:6686–6691.
[Abstract/Free Full Text] - Hinz B, Gabbiani G. Cell–matrix and cell–cell contacts of myofibroblasts: role in connective tissue remodeling. Thromb Haemost (2003) 90:993–1002.[Web of Science][Medline]
- Tomasek JJ, Gabbiani G, Hinz B, et al. Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol (2002) 3:349–363.[CrossRef][Web of Science][Medline]
- Zeisberg M, Hanai J, Sugimoto H, et al. BMP-7 counteracts TGF-b1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med (2003) 9:964–968.[CrossRef][Web of Science][Medline]
- Phanish MK, Wahab NA, Colville-Nash P, et al. The differential role of Smad2 and Smad3 in the regulation of pro-fibrotic TGFb1 responses in human proximal-tubule epithelial cells. Biochem J (2006) 393:601–607.[CrossRef][Web of Science][Medline]
- Valcourt U, Kowanetz M, Niimi H, et al. TGF-b and the Smad signaling pathway support transcriptomic reprogramming during epithelial–mesenchymal cell transition. Mol Biol Cell (2005) 16:1987–2002.
[Abstract/Free Full Text] - Bhowmick NA, Ghiassi M, Bakin A, et al. Transforming growth factor-b1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism. Mol Biol Cell (2001) 12:27–36.
[Abstract/Free Full Text] - Masszi A, Di Ciano C, Sirokmany G, et al. Central role for Rho in TGF-b1-induced alpha-smooth muscle actin expression during epithelial–mesenchymal transition. Am J Physiol Renal Physiol (2003) 284:F911–F924.
[Abstract/Free Full Text] - Rhyu DY, Yang Y, Ha H, et al. Role of reactive oxygen species in TGF-b1-induced mitogen-activated protein kinase activation and epithelial–mesenchymal transition in renal tubular epithelial cells. J Am Soc Nephrol (2005) 16:667–675.
[Abstract/Free Full Text] - Bhowmick NA, Zent R, Ghiassi M, et al. Integrin b 1 signaling is necessary for transforming growth factor-b activation of p38MAPK and epithelial plasticity. J Biol Chem (2001) 276:46707–46713.
[Abstract/Free Full Text] - Ono K, Han J. The p38 signal transduction pathway: activation and function. Cell Signal (2000) 12:1–13.[CrossRef][Web of Science][Medline]
- Pramanik R, Qi X, Borowicz S, et al. p38 isoforms have opposite effects on AP-1-dependent transcription through regulation of c-Jun. The determinant roles of the isoforms in the p38 MAPK signal specificity. J Biol Chem (2003) 278:4831–4839.
[Abstract/Free Full Text] - Fan JM, Ng YY, Hill PA, et al. Transforming growth factor-b regulates tubular epithelial–myofibroblast transdifferentiation in vitro. Kidney Int (1999) 56:1455–1467.[CrossRef][Web of Science][Medline]
- Garat C, Van Putten V, Refaat ZA, et al. Induction of smooth muscle alpha-actin in vascular smooth muscle cells by arginine vasopressin is mediated by c-Jun amino-terminal kinases and p38 mitogen-activated protein kinase. J Biol Chem (2000) 275:22537–22543.
[Abstract/Free Full Text] - Masszi A, Fan L, Rosivall L, et al. Integrity of cell–cell contacts is a critical regulator of TGF-beta 1-induced epithelial-to-myofibroblast transition: role for beta-catenin. Am J Pathol (2004) 165:1955–1967.
[Abstract/Free Full Text] - von Gersdorff G, Susztak K, Rezvani F, et al. Smad3 and Smad4 mediate transcriptional activation of the human Smad7 promoter by transforming growth factor beta. J Biol Chem (2000) 275:11320–11326.
[Abstract/Free Full Text] - Hale KK, Trollinger D, Rihanek M, et al. Differential expression and activation of p38 mitogen-activated protein kinase alpha, beta, gamma, and delta in inflammatory cell lineages. J Immunol (1999) 162:4246–4252.
[Abstract/Free Full Text] - Junttila MR, Ala-Aho R, Jokilehto T, et al. p38alpha and p38delta mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells. Oncogene (2007) 26:5267–5279.[CrossRef][Web of Science][Medline]
- Leivonen SK, Chantry A, Häkkinen L, et al. Smad3 mediates transforming growth factor-beta-induced collagenase-3 (matrix metalloproteinase-13) expression in human gingival fibroblasts. Evidence for cross-talk between Smad3 and p38 signaling pathways. J Biol Chem (2002) 277:46338–46346.
[Abstract/Free Full Text] - Davies SP, Reddy H, Caivano M, et al. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J (2000) 351:95–105.[CrossRef][Web of Science][Medline]
- Bakin AV, Rinehart C, Tomlinson AK, et al. p38 mitogen-activated protein kinase is required for TGFbeta-mediated fibroblastic transdifferentiation and cell migration. J Cell Sci (2002) 115:3193–3206.
[Abstract/Free Full Text] - Tourian L Jr, Zhao H, Srikant CB. p38alpha, but not p38beta, inhibits the phosphorylation and presence of c-FLIPS in DISC to potentiate Fas-mediated caspase-8 activation and type I apoptotic signaling. J Cell Sci (2004) 117:6459–6471.
[Abstract/Free Full Text] - Hu B, Wu Z, Phan SH. Smad3 mediates transforming growth factor-beta-induced alpha-smooth muscle actin expression. Am J Respir Cell Mol Biol (2003) 29:397–404.
[Abstract/Free Full Text] - Hayashida T, Decaestecker M, Schnaper HW. Cross-talk between ERK MAP kinase and Smad signaling pathways enhances TGF-beta-dependent responses in human mesangial cells. Faseb J (2003) 17:1576–1578.
[Abstract/Free Full Text] - Takekawa M, Tatebayashi K, Itoh F, et al. Smad-dependent GADD45beta expression mediates delayed activation of p38 MAP kinase by TGF-beta. Embo J (2002) 21:6473–6482.[CrossRef][Web of Science][Medline]
- Ungefroren H, Lenschow W, Chen WB, et al. Regulation of biglycan gene expression by transforming growth factor-beta requires MKK6-p38 mitogen-activated protein kinase signaling downstream of Smad signaling. J Biol Chem (2003) 278:11041–11049.
[Abstract/Free Full Text] - Leivonen SK, Ala-Aho R, Koli K, et al. Activation of Smad signaling enhances collagenase-3 (MMP-13) expression and invasion of head and neck squamous carcinoma cells. Oncogene (2006) 25:2588–2600.[CrossRef][Web of Science][Medline]
Accepted in revised form: 10.10.07
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